Volume 47, Issue 5, Pages 903-912.e4 (November 2017) The Cytokine TGF-β Promotes the Development and Homeostasis of Alveolar Macrophages  Xueyang Yu, Anne Buttgereit, Iva Lelios, Sebastian G. Utz, Dilay Cansever, Burkhard Becher, Melanie Greter  Immunity  Volume 47, Issue 5, Pages 903-912.e4 (November 2017) DOI: 10.1016/j.immuni.2017.10.007 Copyright © 2017 Elsevier Inc. Terms and Conditions

Immunity 2017 47, 903-912.e4DOI: (10.1016/j.immuni.2017.10.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 TGF-β Is Required for AMs in an Autocrine Manner (A–C) Flow cytometry of lung cells derived from ItgaxCreTgfbr2fl/fl mice and control littermates (Tgfbr2fl/fl or ItgaxCreTgfbr2fl/+) at postnatal day (P)28 and P14. Representative flow cytomety plots and graphs (±SEM) show the frequency and total cell numbers of AMs (Siglec-F+CD11c+, pre-gated on CD45+ cells) (A) at P14 and P28 and CD103+ DCs, CD11b+ DCs and interstitial macrophages (IMF) (pre-gated on CD45+Ly6G−Siglec-F−Ly6C−MHCII+CD11c+) at P14 (C). N = 4-11 from 2-3 independent experiments (A) and N = 5 from 2 independent experiments (C). (B) Annotated t-SNE plots and expression of markers in the identified populations among CD45+ lung cells from Tgfbr2fl/fl or ItgaxCreTgfbr2fl/fl mice (P14) as in (A). Archsinh transformed medians are shown. (D) ELISA for TGF-β1 of the lung, liver, kidney and brain from WT mice, normalized to total protein amount. N = 3. (E) Relative mRNA expression of Tgfb1 from sorted AMs, epithelial cells (EpC, pre-gated on CD45−Epcam+CD31−), endothelial cells (EnC, pre-gated on CD45−Epcam−CD31+) and other CD45− cells from WT lungs, normalized to Pol2. N ≥ 3. (F-G) Representative flow cytometry plots of AMs (Siglec-F+CD11c+) among CD45+ cells from lung tissue (F) or from bronchoalvelar lavage (BAL) (G) from ItgaxCreTgfb1fl/fl mice and control littermates (Tgfb1fl/fl, Tgfb1fl/+ or ItgaxCreTgfb1fl/+) and quantification of total cell numbers (±SEM) on the right. N ≥ 4 from 2 independent experiments. (H) Oil Red O staining of AMs derived from the BAL of ItgaxCreTgfb1fl/fl mice and from control littermates. Scale bar = 20 μm. Representative images are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant (unpaired Student’s t test for (A and C) and (F-G), one-way ANOVA for (D-E)). See also Figures S1 and S2. Immunity 2017 47, 903-912.e4DOI: (10.1016/j.immuni.2017.10.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 TGF-βR Signaling Is Required for the Homeostasis of AMs (A and B) R26CreERTgfbr2fl/fl mice and Tgfbr2fl/fl littermates were treated with tamoxifen (5 mg) 5 times every other day via oral gavage (o.g.) and analyzed 7 days after the last treatment. (A) Flow cytometry (left) of AMs (Siglec-F+CD11c+, pre-gated on CD45+ cells) and quantification (right) of total cell numbers (±SEM). N ≥ 9 from 4 independent experiments. (B) qRT-PCR of Tgfbr2 mRNA from AMs (Siglec-F+CD11c+) as shown in (A), normalized to Pol2 expression. N = 3. (C–E) Cx3cr1CreERTgfbr2fl/fl mice and Tgfbr2fl/fl littermates were treated with tamoxifen every other day (max. 5 times) via o.g. and analyzed 6 and 14 days after treatment start. Representative flow cytometry plots (left) on D6 and quantification of total cell numbers (right) on D6 and D14 of kidney macrophages (MF) (C) and small intestinal lamina propria macrophages (SI MF) (D) (pre-gated on CD45+Ly6G−Siglec-F−Ly6C−MHCII+CD11c+ cells). N ≥ 3 from 2-8 independent experiments. (E) qRT-PCR of Tgfbr2, Il1b and Tnf mRNA from kidney MF and SI MF at D6, normalized to Pol2 expression. N = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant (unpaired Student’s t test). Immunity 2017 47, 903-912.e4DOI: (10.1016/j.immuni.2017.10.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 TGF-βR Signaling Is Indispensable for the Development of AMs during Embryogenesis (A) Representative flow cytometry plots (left) and quantification of total cell numbers (±SEM) (right) of fetal monocytes (fetal Mo, Ly6ChiCD11bhi) and preAMs in Lyz2CreTgfbr2fl/fl and Lyz2CreTgfbr2fl/+ (homozygous for Lyz2Cre) embryos at E18.5 (pre-gated on CD45+Ly6G−MHCII−F4/80loCD11b+CD64+ cells). N = 13, 4 independent experiments. (B) Relative mRNA expression (±SEM) of Tgfbr2 from sorted fetal monocytes and preAMs as shown in (A) normalized to Pol2. N = 3, each from 2-4 pooled mice. (C) Flow cytometry analysis of fetal lung of Vav1CreTgfbr2fl/fl and Tgfbr2fl/fl or Vav1CreTgfbr2fl/+ control mice at E18.5. Total cell numbers (±SEM) are quantified on the right. N ≥ 5, from 2-3 independent experiments. (D) Fold change of relative mRNA expression of Tgfbr2, Pparg, Csf2rb, Tgfb1 and Car4 from E15.5 fetal liver monocytes cultured in vitro with TGF-β1 or with anti-TGF-β1 blocking antibodies, normalized to Pol2. N = 3 from 3 independent experiments. (E) Relative mRNA expression levels of Pparg, Car4, Spi1, Csf2rb, Csf2ra, Tgfbr1 and Tgfb1 from sorted fetal monocytes and preAMs as described in (A-B), normalized to Pol2. N ≥ 3, each from 2-4 pooled mice. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant (unpaired Student’s t test). See also Figure S1 and S4. Immunity 2017 47, 903-912.e4DOI: (10.1016/j.immuni.2017.10.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Gene expression profiles of Tgfbr2-deficient AMs (A) Flow cytometry plots show the percentage of AMs (Siglec-F+CD11c+, pre-gated on CD45+ cells) of ItgaxCreTgfbr2fl/fl and Tgfbr2fl/fl mice at P3. (B-H) AMs were sorted from ItgaxCreTgfbr2fl/fl and Tgfbr2fl/fl mice at P3 as in (A) for NGS. (See also gating strategy for NGS in Figure S1C). N = 4, each from 2-4 mice. (B) Normalized expression counts of Exon 4 of Tgfbr2. (C) Heat map of expression values of genes expressed differentially in ItgaxCreTgfbr2fl/fl or Tgfbr2fl/fl AMs. (Significant p value < 0.001, FDR = 0.020). (D) Heat map showing differentially expressed genes belonging to the TGF-βR signaling pathway (GO:0007179, log10P = -5.25, fold change (FC)). (E) GSEA of genes expressed differentially in ItgaxCreTgfbr2fl/fl compared to Tgfbr2fl/fl AMs. Enrichment plot for ‘TGF-β signaling’. (P = 0.0045, FDR= 0.034). (F) Differentially expressed AM and monocyte signature genes in ItgaxCreTgfbr2fl/fl and Tgfbr2fl/fl AMs (log2 (FC) > 0.5) (Gautier et al., 2012). (G) Fold change in expression of the 25 most significantly up- or down-regulated genes in AMs from ItgaxCreTgfbr2fl/fl mice compared to Tgfbr2fl/fl mice. (H) Normalized expression counts of Pparg, Runx1, Csf1r, Csf2rb, Apoe and Spi1. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant (unpaired Student’s t test). See also Figure S1. Immunity 2017 47, 903-912.e4DOI: (10.1016/j.immuni.2017.10.007) Copyright © 2017 Elsevier Inc. Terms and Conditions